Method of analyzing ligand in sample and apparatus for analyzing ligand in sample

ABSTRACT

A sample analyzing method capable of high-precision analysis without being affected by vibration and optical design is provided. In the sample analyzing method, a sample containing a ligand is caused to bind to a receptor that can bind specifically to the ligand, and a change caused by binding of the receptor and the ligand is analyzed by measuring frequency characteristics of a surface plasmon resonance angle while applying external vibration to the receptor. The sample analyzing method is useful in the fields of, for example, biology, medicine, pharmacology, agriculture, and the like.

TECHNICAL FIELD

The present invention relates to a method for analyzing a ligand in asample and an apparatus for analyzing a ligand in a sample.

BACKGROUND ART

Among biochemical reactions, the reaction of binding a receptor and aligand is important. For example, in an antigen-antibody reaction, anantibody as a receptor binds to an antigen as a ligand. For example, inan enzymatic reaction, an enzyme as a receptor binds to an enzymesubstrate as a ligand. By utilizing these reactions, a ligand present ina sample can be detected.

For example, a receptor is bound to a metal thin film surface. When aligand binds to the receptor, the dielectric constant in the vicinity ofthe metal thin film is changed. A method of using a surface plasmonresonance phenomenon to detect such a change and thereby analyze theamount of a receptor-ligand complex, has been known (see, for example,Non-patent document 1). In this method, initially, a metal thin film towhose surface a receptor is bound as described above is irradiated fromthe rear side with light at an angle that satisfies a total internalreflection condition. When the angle is a specific incident angle thatcauses the wave number of a surface plasmon excited by the incidentlight to be equal to the wave number of an evanescent wave derived fromthe excitation light, a portion of the amount of the incident light isused for excitation of the surface plasmon, so that the amount ofreflected light is reduced. For example, in order to detect thedielectric constant change in the vicinity of the metal thin film, amethod of measuring reflected light while changing the angle of incidentlight to determine an angle of incident light at which absorption ishighest, and a method of determining an angle of reflected light atwhich absorption is highest where the angle of incident light is heldconstant, have been known. In addition, a technique of applying anelectric field (electrical vibration) to the rear side of a metal thinfilm on which a receptor is immobilized to control separation andmovement of a sample on the metal thin film (see, for example, Patentdocument 1) and a technique of using a change in refractive index of ametal thin film to measure a large amount of sample at a time (see, forexample, Patent document 2), have been proposed.

However, in these methods, the amount of a ligand binding to a receptoris analyzed based on a minute amount of angle change corresponding to areduction in light amount due to excitation of surface plasmon.Therefore, in these methods, the surface of a metal thin film (e.g.,thickness: about several nanometers) on which incident light isreflected needs to be even. In addition, since a minute angle changeamount is measured based on a change in light amount, optical parts needto be adjusted with high precision so that light can be received withoutdeviation of an optical axis. As a result, apparatuses for use in thesemethods tend to be influenced by optical design. In addition, since aminute change is detected, reading precision deteriorates significantlywhen an apparatus suffers from vibration during measurement. Therefore,it is difficult to produce portable apparatuses that are suitable forthese methods.

-   Non-patent document 1: Anal. Chem., 1998, 70,2019-2024-   Patent document 1: JP 2003-65947 A-   Patent document 2: JP 2003-75336 A

DISCLOSURE OF INVENTION

Problem to be Solved by the Invention

An object of the present invention is to provide a method and anapparatus for analyzing a ligand in a sample with high precision thatare less susceptible to vibration and optical design compared withconventional techniques.

Means for Solving Problem

In order to achieve the object, the present invention provides a methodfor analyzing a ligand in a sample, comprising the steps of providing asample containing a ligand, providing a metal thin film, wherein areceptor that can bind specifically to a ligand is immobilized on oneside of the metal thin film, an optical prism is provided on an oppositeside of the metal thin film, and the metal thin film can cause surfaceplasmon resonance, providing irradiating means for irradiating withmeasuring light, providing light receiving means for receiving reflectedlight of the measuring light, providing analyzing means for analyzing aligand binding to the receptor, causing the sample and the metal thinfilm to contact each other so that the ligand in the sample binds to thereceptor, irradiating the side of the metal thin film opposite to theside on which the receptor is immobilized with measuring light using theirradiating means, receiving reflected light of the measuring lightreflected on the side of the metal thin film using the light receivingmeans, and detecting a change in a surface plasmon resonance anglecaused by a change in a dielectric constant of a vicinity of the metalthin film, based on the reflected light, using the analyzing means,further comprising providing applying means for applying externalvibration to a region in which the receptor is immobilized, applyingexternal vibration to the side of the metal thin film on which thereceptor is immobilized, using the applying means, while irradiating themetal thin film with the measuring light using the irradiating means,and obtaining frequency characteristics of a surface plasmon resonanceangle with respect to external vibration using the analyzing means, andbased on the frequency characteristics, analyzing a ligand in the samplebinding to the receptor.

The present invention also provides an apparatus for analyzing a ligandin a sample, comprising a metal thin film, wherein a receptor that canbind specifically to a ligand is immobilized on one side of the metalthin film, an optical prism is provided on an opposite side of the metalthin film, and the metal thin film can cause surface plasmon resonance,irradiating means for irradiating with measuring light, light receivingmeans for receiving reflected light of the measuring light reflected onthe side of the metal thin film, and analyzing means for analyzing aligand binding to the receptor, and further applying means for applyingexternal vibration to the side of the metal thin film on which thereceptor is immobilized. A side of the metal thin film opposite to theside on which the receptor is immobilized can be irradiated withmeasuring light using the irradiating means while applying externalvibration using the applying means, and the analyzing means can detect achange in a surface plasmon resonance angle from the reflected light andobtain frequency characteristics of a surface plasmon resonance anglewith respect to external vibration, and based on the frequencycharacteristics, analyze a ligand in the sample binding to the receptor.

Effects of the Invention

As described above, in the analyzing method and apparatus of the presentinvention, external vibration is applied, and the frequencycharacteristics of a surface plasmon resonance angle with respect to theexternal vibration are obtained. Due to the external vibration, adielectric constant in a vicinity of the metal thin film is changed. Thedielectric constant change causes a change in the surface plasmonresonance angle. Thus, the analyzing method and apparatus of the presentinvention measures the frequency characteristics of the surface plasmonresonance angle. Therefore, it is not necessary to measure a minutechange in the surface plasmon resonance angle. As a result, it ispossible to achieve high-precision analysis without being affectedsignificantly by vibration and optical design. In addition, theapparatus of the present invention can resist vibration, and therefore,can be miniaturized.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating an exemplary apparatus of thepresent invention.

FIG. 2 is a diagram for explaining a method for evaluating a physicalproperty by applying electrical vibration as external vibration, in anexemplary apparatus of the present invention. FIG. 2( a) is a diagramillustrating frequency characteristics of an impedance of a ligand thatwere measured. FIG. 2( b) is a schematic diagram in which a ligand isrepresented by a combination of equivalent circuits.

FIG. 3 illustrates examples of a surface plasmon resonance curvemeasured when no receptor was provided on a metal thin film, and asurface plasmon resonance curve measured when water was provided as areceptor, in an exemplary apparatus of the present invention. FIG. 3( a)illustrates the surface plasmon resonance curve in the absence of areceptor, and FIG. 3( b) illustrates the surface plasmon resonance curvewhen water is used as a receptor.

FIG. 4A illustrates exemplary surface plasmon resonance curves measuredwithout a ligand, in the exemplary apparatus of the present invention.FIG. 4A(a) illustrates a surface plasmon resonance curve in the absenceof a ligand, and FIG. 4A(b) is a partially enlarged view of FIG. 4A(a).

FIG. 4B illustrates exemplary surface plasmon resonance curves measuredusing a ligand, in the exemplary apparatus of the present invention.FIG. 4B(a) illustrates a surface plasmon resonance curve when albuminbovine serum was used as a ligand, and FIG. 4B(b) illustrates apartially enlarged view of FIG. 4B(a).

FIG. 5 illustrates a schematic diagram indicating exemplary states of avicinity of a metal thin film depending on the intensity of externalvibration, in the apparatus of the present invention. FIG. 5( a) is aschematic diagram illustrating a state when the intensity of externalvibration is zero, FIG. 5( b) is a schematic diagram illustrating astate when the intensity of external vibration is weak, and FIG. 5( c)is a schematic diagram illustrating a state when the intensity ofexternal vibration is strong.

FIG. 6 illustrates a diagram for explaining a relationship betweenexternal vibration and binding of a receptor and a ligand in the presentinvention. FIG. 6( a) is a diagram illustrating a temporal change inapplied external vibration. FIG. 6( b) is a diagram illustrating atemporal change in amount of a ligand-receptor complex. FIG. 6( c) is adiagram illustrating a sum of a phase of applied external vibration anda phase of reflected light that causes plasmon resonance. FIG. 6( d) isa diagram illustrating a digital signal that is obtained by conversionof an analog signal of FIG. 6( c) using an A/D converter.

FIG. 7 is a schematic diagram illustrating another exemplary apparatusof the present invention.

DESCRIPTION OF THE INVENTION

In the analyzing method and apparatus of the present invention, at leastone of a receptor and a ligand preferably is charged. This is because,in this case, the dielectric constant of the metal thin film surface canbe easily changed when electrical vibration is applied as externalvibration. Examples of a combination of a receptor and a ligand includean antigen and an antibody; an antibody and an antigen; a hormone and ahormone receptor; a hormone receptor and a hormone; a polynucleotide anda polynucleotide receptor; a polynucleotide receptor and apolynucleotide; an enzyme inhibitor and an enzyme; an enzyme and anenzyme inhibitor; an enzyme substrate and an enzyme; an enzyme and anenzyme substrate; and the like.

The present inventors infer a mechanism of the dielectric constantchange in the vicinity of the metal thin film as follows. When externalvibration is applied to the receptor and the ligand, the receptor andthe ligand follow the metal thin film and gather close together on themetal thin film surface. As a result, the molecular density of anevanescent region on the metal thin film is changed. The moleculardensity change is responsible for a change in the dielectric constant ofthe evanescent region, i.e., the dielectric constant change in thevicinity of the metal thin film.

In the analyzing method of the present invention, the applying meanspreferably is means for applying at least one of electrical vibration,magnetic vibration, and mechanical vibration, more preferably, means forapplying at least electrical vibration. In this case, the applying meansis means for applying at least electrical vibration, and the analyzingmeans preferably further includes analyzing a physical property of theligand from the reflected light. This is because, in this case, thephysical property of the ligand can be easily analyzed. Similarly, inthe apparatus of the present invention, the applying means preferably ismeans for applying at least one of electrical vibration, magneticvibration, and mechanical vibration, more preferably means for applyingat least electrical vibration. In this case, the applying means is meansfor applying at least electrical vibration, and the analyzing meanspreferably can analyze a physical property of the ligand from thereflected light. This is because, in this case, the physical property ofthe ligand, the analysis of which conventionally requires anotherapparatus, can be analyzed simultaneously. The electrical vibrationfurther preferably is provided by using an alternating current electricfield. This is because, by applying alternating current electric field,an electrochemical property, such as a charge number, an electricalresistance value, or the like, of at least one of the ligand and thereceptor can be evaluated simultaneously.

In the analyzing method of the present invention, an amount of a ligandin the sample binding to the receptor preferably is analyzed. Similarly,in the apparatus of the present invention, an amount of a ligand in thesample binding to the receptor preferably is analyzed.

In the analyzing method of the present invention, preferably, theanalyzing means further comprises comparing means for comparing a phaseof the external vibration with a phase of a signal component of theexternal vibration included in the reflected light, and the step ofobtaining the frequency characteristics uses the comparing means tocompare the phase of the external vibration with the phase of the signalcomponent of the external vibration included in the reflected light, todetect a point of inflection (following frequency limit) of thefrequency characteristics. This is because, by detecting the point ofinflection of the frequency characteristics, a result having highermeasurement precision can be obtained than that of a method of measuringa change in a surface plasmon resonance angle. Similarly, in theapparatus of the present invention, the analyzing means furthercomprises comparing means capable of comparing a phase of the externalvibration with a phase of a signal component of the external vibrationincluded in the reflected light, to detect a point of inflection(following frequency limit) of the frequency characteristics. This isbecause, by detecting the point of inflection of the frequencycharacteristics, a result having higher measurement precision can beobtained than when a change in a surface plasmon resonance angle ismeasured.

Preferably, the analyzing method of the present invention furthercomprises providing measuring means for measuring a temporal change inthe point of inflection of the frequency characteristics, and bymeasuring the temporal change in the point of inflection of thefrequency characteristics using the measuring means, the degree ofbinding of the receptor and the ligand is detected. This is because, inthis case, the degree of the progress of binding of the receptor and theligand can be measured. Similarly, it is preferable that in theapparatus of the present invention, measuring means is further included,and by measuring a temporal change in the point of inflection of thefrequency characteristics using the measuring means, the degree ofbinding of the receptor and the ligand can be detected. This is because,in this case, the degree of the progress of binding of the receptor andthe ligand can be measured.

Preferably, the analyzing method of the present invention furthercomprises providing optical means for causing reflected light of themeasuring light reflected on the side of the metal thin film on whichthe receptor is immobilized to impinge on the side further a pluralityof times, and the optical means is used to cause reflected light of themeasuring light reflected on the side of the metal thin film on whichthe receptor is immobilized, to impinge on the side further a pluralityof times, and the reflected light of the measuring light received by thelight receiving means is the reflected light of the measuring lightreflected a plurality of times on the side of the metal thin film usingthe optical means. This is because, in this case, a plurality of timesof reflection amplifies the amplitude of the surface plasmon resonanceangle to achieve high-sensitivity measurement. Similarly, it ispreferable that the apparatus of the present invention further comprisesoptical means capable of causing the reflected light of the measuringlight reflected on the side of the metal thin film on which the receptoris immobilized, to impinge on the side further a plurality of times, andthe reflected light of the measuring light received by the lightreceiving means is the reflected light of the measuring light reflecteda plurality of times on the side of the metal thin film using theoptical means. This is because, in this case, a plurality of times ofreflection amplifies the amplitude of the surface plasmon resonanceangle to achieve high-sensitivity measurement.

First Embodiment

In a first embodiment of the present invention, a preferable embodimentof the apparatus of the present invention will be described. FIG. 1 is aschematic diagram illustrating an example of the apparatus of thepresent invention. In FIG. 1, 1 indicates a light source, 2 indicates aprism, 3 indicates measuring light. 4 indicates a light receivingapparatus. 5 indicates reflected light. 6 indicates a reflected lightdark portion in which the amount of light is partially reduced due toexcitation of plasmon. 7 indicates a metal thin film and upperelectrode. 8 indicates a lower electrode. 9 indicates a receptorimmobilized on the metal film and upper electrode 7. 10 indicates analternating current source. 11 indicates a frequency divider thatdivides a frequency of the alternating current source 10. 12 and 16 eachindicate an active filter that passes only a specific frequency. 13indicates external vibration. 14 indicates a capacitor. 15 indicates anamplifier. 17 indicates a detector that compares a phase (θ₀) ofexternal vibration with a phase (θ₁) of a signal component of externalvibration included in reflected light. 18 indicates an A/D converterthat converts an analog signal obtained by the detector 17 into adigital signal. 39 indicates a ligand, and θ indicates an incidentangle.

The light source 1 is provided at a location that allows the measuringlight 3 emitted by the light source 1 to impinge on the metal thin filmand upper electrode 7. The receptor 9 is immobilized on a side oppositeto an irradiated side of the metal thin film and upper electrode 7 thatis irradiated. The light receiving apparatus 4 is provided at a locationthat allows the light receiving apparatus 4 to receive the reflectedlight 5 from the metal thin film and upper electrode 7. The lowerelectrode 8 is disposed so that external vibration can be applied to thereceptor 9 immobilized on the metal thin film and upper electrode 7. Thelower electrode 8 is connected via the frequency divider 11 and theactive filter 12 to the alternating current source 10, and thealternating current source 10 also is connected to the other activefilter 16. The active filter 16 is connected via the capacitor 14 andthe amplifier 15 in this order to the light receiving apparatus 4. Boththe active filter 16 and the active filter 12 are connected via thedetector 17 to the A/D converter 18. In FIG. 1, the metal thin film andupper electrode 7 is provided in an upper portion of the apparatus,while the lower electrode 8 is provided in a lower portion of theapparatus. Alternatively, the positional upper-lower relationship may bereversed, or the electrodes may be provided effectively on right andleft sides of the apparatus.

The measuring light 3 emitted by the light source 1 is passed through abeam splitter (not shown) to extract p-polarized light, and only thep-polarized light is passed through the prism 2 (e.g., a prismmanufactured by Nippon Denshi Reza) while changing the incident angle θand is brought onto the metal thin film and upper electrode 7. Themeasuring light 3 incident to the metal thin film and upper electrode 7is reflected totally on the metal thin film and upper electrode 7 togenerate the reflected light 5. When the measuring light 3 is caused toenter the metal thin film and upper electrode 7 at a certain particularincident angle θ, on evanescent wave is generated, so that a portion ofthe light amount is used for excitation of plasmon wave that is calledsurface plasmon resonance, and a reflected light dark portion 6 having areduced light amount is generated. The light receiving apparatus 4 thatconverts an intensity of the reflected light 5 into a voltage is used todetect the intensity of the reflected light 5 including the reflectedlight dark portion 6.

Further, a unit composed of the alternating current source 10 and thefrequency divider 11 is connected to the metal thin film and upperelectrode 7 and the lower electrode 8. The alternating current source 10generates a frequency of nf, which is in turn converted into a frequencyof f by the frequency divider 11. The lower electrode 8 applies theexternal vibration 13 having a frequency of f to the metal thin film andupper electrode 7. As a result, the receptor 9 is vibrated due to achange in direction of the molecule. The electronic polarizationcomponent of the receptor molecule is dominantly responsible for thedielectric constant of an evanescent wave region with respect to themeasuring light. When a direction of the receptor 9 in an evanescentregion of the surface of the metal thin film and upper electrode 7 ischanged due to the external vibration 13, a center of electron densityalso is changed, so that the dielectric constant is changed, and theparticular angle of the reflected light dark portion 6 at which plasmonresonance is generated is changed within a range including the lightreceiving apparatus 4.

When the ligand 39 binds to the receptor 9 in the vicinity of the metalthin film and upper electrode 7, the weight of the receptor 9 isincreased by the molecular weight of the ligand 39. As a result, thedielectric constant of a complex of the receptor 9 and the ligand 39 ischanged from the dielectric constant of only the receptor 9. When theexternal vibration 13 is applied to the receptor 9 to which the ligand39 binds, the frequency characteristics of vibration are changed due tothe direction change of the receptor 9 with respect to the externalvibration 13.

The reflected light 5 including the reflected light dark portion 6 isdetected by the light receiving apparatus 4, and the alternating currentcomponent is passed through and amplified by the amplifier 15. Theactive filter 16 removes components other than a signal component ofexternal vibration. The detector 17 compares the phase (θ₀) of externalvibration with the phase (θ₁) of a signal component of externalvibration included in reflected light. The obtained analog signal isconverted into a digital signal by the A/D converter 18.

In the apparatus of the present invention, by measuring the frequencycharacteristics of the surface plasmon resonance angle, it is possibleto analyze a ligand binding to a receptor, for example, it is possibleto analyze an amount of a ligand binding to a receptor. Thus, in theapparatus of the present invention, it is not necessary to read a minutechange in angle of the reflected light dark portion 6, whereby theoptical flatness of a measuring instrument, such as the prism 2, themetal thin film and upper electrode 7, or the like, is relaxed. Further,in the apparatus of the present invention, the light receiving apparatus4 may be provided at a location within a change range of the reflectedlight dark portion 6, so that the influence of noise is reduced, therebymaking it possible to achieve high-sensitivity measurement.

Since the optical flatness of a measuring instrument, the precision ofparts, and the precision of an optical axis are relaxed, the apparatusof the present invention can be manufactured with low cost and can beprovided as a portable and small-size apparatus that resists vibration.Further, by applying electrical vibration as external vibration, aphysical property of a ligand that conventionally cannot be evaluatedsimultaneously, such as a charge number, an electrical resistance value,or the like, can be evaluated. For example, a predetermined voltage isapplied between the metal thin film and upper electrode 7 and the lowerelectrode 8 while changing a frequency generated from the alternatingcurrent source 10, and by measuring a current flowing at that time,frequency characteristics of impedance is obtained as illustrated inFIG. 2( a). A ligand contained in a sample can be represented by acombination of equivalent circuits, such as a resistance (R), acapacitance (C), and the like, as illustrated in FIG. 2( b). Impedance Zis Z=R+jX in the case of series arrangement, andZ=RX²/(R²+X²)+jR²X/(R²+X²) in the case of parallel arrangement (in theexpressions, X=−1/(2πfC)). For example, these expressions are applied tothe impedance-frequency characteristics of FIG. 2( a) for analysis,thereby making it possible to evaluate each property. Note that themethod of applying a predetermined voltage between the metal thin filmand upper electrode 7 and the lower electrode 8 while changing afrequency generated by the alternating current source 10, and analyzinga current flowing at that time has been described. This method may beachieved by applying a predetermined voltage between the metal thin filmand upper electrode 7 and the lower electrode 8 while changing afrequency generated by the alternating current source 10, and measuringa voltage applied at that time. Note that, in FIG. 2( b), R₁, R₂, and R₃indicate a resistance in a ligand, a resistance between ligands, and aresistance of an electrode, respectively, and C₁, C₂, and C₃ indicate acapacitance in a ligand, a capacitance between ligands, and acapacitance of an electrode, respectively.

Note that, as the light source 1, an He—Ne laser, an Ar laser, a pigmentlaser, or the like can be used. For example, the light source 1 may becomposed of an AlGaAs double heterojunction visible light semiconductorlaser (e.g., manufactured by ROHM Co., Ltd.), a collimating lens (forexample, Panasonic Electronic Devices Nitto Co., Ltd.), and a polarizedbeam splitter (for example, manufactured by Sigma Koki Co., Ltd.). As amethod for changing the incident angle, the light source 1 may be drivento move the measuring light 3 to scan the metal thin film and upperelectrode 7, or alternatively, the light source 1 is fixed and areflection mirror, such as a polygon mirror scanner or the like, isdriven to move the measuring light 3 for scanning.

The prism 2 can be in the shape of a cone, a hemisphere, or the like.

The metal thin film and upper electrode 7 is not limited as long as itcan cause surface plasmon resonance. For example, the metal thin filmand upper electrode 7 can be composed of a metal thin film made ofplatinum, gold, or the like, preferably a gold metal thin film (e.g.,manufactured by Nippon Denshi Reza). As the receptor 9, those describedabove can be used. For example, the receptor is immobilized on a metalthin film directly, or indirectly with a binding modification molecule,thereby producing the metal thin film on one side of which the receptoris immobilized.

The light receiving apparatus 4 may be an apparatus that converts theintensity of the reflected light 5 into a voltage using a CCD, an Si PINphotodiode (e.g., manufactured by Hamamatsu Photonics, K. K.), anoperational amplifier (e.g., manufactured by National SemiconductorCorporation), a resistance device, and the like.

FIGS. 3( a) to 3(b) illustrate examples of a surface plasmon resonancecurve measured when no receptor was provided on a metal thin film, and asurface plasmon resonance curve measured when water was provided as areceptor, in the example of the apparatus of the present invention. Ineach graph, the horizontal axis indicates the incident angle θ of themeasuring light 3, and the vertical axis indicates the intensity of thereflected light 5. FIG. 3( a) illustrates the surface plasmon resonancecurve in the absence of a receptor, and FIG. 3( b) illustrates thesurface plasmon resonance curve when water is used as a receptor. Notethat, in this case, external vibration is not applied duringmeasurement. As illustrated in FIGS. 3( a) and 3(b), an angle thatcauses surface plasmon resonance varies depending on the presence orabsence of a receptor. In other words, it is illustrated that theexemplary apparatus of the present invention can measure a change in thesurface plasmon resonance angle associated with a change in thedielectric constant in the vicinity of a metal thin film.

FIGS. 4A(a), 4A(b), 4B(a), and 4B(b) illustrate a surface plasmonresonance curve of a sample without a ligand and a surface plasmonresonance curve of a sample containing a ligand, in the exemplaryapparatus of the present invention. In each graph, the horizontal axisindicates the incident angle θ of the measuring light 3, and thevertical axis indicates the intensity of the reflected light 5.

As a measuring apparatus, the exemplary apparatus of the presentinvention of FIG. 1 was used. As a receptor, anti-albumin bovine serumantibodies (manufactured by Sigma-Aldrich, Inc.) was used. As a sample,1% phosphate buffer solution that was prepared by adding sodiumhydroxide to phosphoric acid (manufactured by Sigma-Aldrich, Inc.) to pH7.0, or 1% phosphate buffer solution that was prepared by adding albuminbovine serum (manufactured by Sigma-Aldrich, Inc.) and phosphoric acid(manufactured by Sigma-Aldrich, Inc.) to sodium hydroxide to 10 μg/mland pH 7.0, respectively, was used. For both the cases when externalvibration was applied and when external vibration was not applied,surface plasmon resonance curves of the samples were obtained. FIG.4A(a) illustrates a surface plasmon resonance curve in the absence of aligand, and FIG. 4A(b) illustrates a partially enlarged view of FIG.4A(a). FIG. 4B(a) illustrates a surface plasmon resonance curve whenalbumin bovine serum was used as a ligand, and FIG. 4B(b) illustrates apartially enlarged view of FIG. 4B(a).

As illustrated in FIGS. 4A(a) and 4A(b), the surface plasmon resonancecurves of the samples without a ligand are substantially the sameregardless of whether or not electric field (external vibration) wasapplied. On the other hand, as illustrated in FIGS. 4B(a) and 4B(b), thesurface plasmon resonance curve of the sample containing a ligand,measured in the presence of applied electric field, is shifted from thesurface plasmon resonance curve measured in the absence of appliedelectric field. The present inventors infer that these results arecaused by a mechanism illustrated in FIGS. 5( a) to 5(c).

FIGS. 5( a) to 5(c) illustrate schematic diagrams indicating exemplarystates of the vicinity of the metal thin film depending on the intensityof external vibration, in the apparatus of the present invention. FIG.5( a) is a schematic diagram illustrating a state when the intensity ofexternal vibration is zero, FIG. 5( b) is a schematic diagramillustrating a state when the intensity of external vibration is weak,and FIG. 5( c) is a schematic diagram illustrating a state when theintensity of external vibration is strong. In each diagram, receptors22, 27, and 33 are immobilized on first sides of metal thin film andupper electrodes 21, 26, and 32, respectively. Measuring light beams 19,24, and 30 are reflected on surfaces of the metal thin film and upperelectrodes 21, 26, and 32, respectively, to generate reflected lightdark portions 20, 25, and 31, respectively. In these cases, respectivesurface plasmon resonance angles are θ₁₁, θ₁₂, and θ₁₃. In FIGS. 5( a)to 5(c), 23, 28, and 34 indicate lower electrodes, and 29 and 35indicate external vibration.

Specifically, when the intensity of external vibration is zero, asillustrated in FIG. 5( a), when a side of the metal thin film and upperelectrode 21 opposite to the side on which the receptor 22 isimmobilized is irradiated with the measuring light 19, the measuringlight 19 is reflected on the surface of the metal thin film and upperelectrode 21, so that the reflected light dark portion 20 occurs. Inthis case, the receptor 22 does not follow the metal thin film and upperelectrode 21, so that the surface plasmon resonance angle is θ₁₁.

When the intensity of external vibration is weak, the receptor 27slightly follows the metal thin film and upper electrode 26, so that thereceptor molecules 27 gather close together on a surface of the metalthin film and upper electrode 26 as illustrated in FIG. 5( b).Therefore, the molecular density of an evanescent region on the metalthin film and upper electrode 26 is changed. Therefore, when a side ofthe metal thin film and upper electrode 26 opposite to the side on whichthe receptor 27 is immobilized is irradiated with the measuring light24, the measuring light 24 is reflected on the surface of the metal thinfilm and upper electrode 26, so that the reflected light dark portion 25occurs. The molecular density of this region is changed, so that thedielectric constant of an evanescent region also is changed, andtherefore, an angle that causes surface plasmon resonance also ischanged. In this case, the surface plasmon resonance angle is θ₁₂.

When the intensity of external vibration is strong, the receptor 33follows the metal thin film and upper electrode 32, so that the receptormolecules 33 gather close together on the surface of the metal thin filmand upper electrode 32 as illustrated in FIG. 5( c). Therefore, an angleof the reflected light dark portion 31 that causes surface plasmonresonance is changed. The surface plasmon resonance angle is θ₁₃. Thus,although surface plasmon resonance occurs at a single angle inconventional surface plasmon measuring apparatuses, the plasmonresonance occurs at a plurality of angles (not one) over time byapplying external vibration. Therefore, a dark line caused by surfaceplasmon resonance has a width. Note that the receptor 9 is released fromthe vicinity of the metal thin film and upper electrode 7 by applyingalternating current electric field, i.e., reversing the direction ofelectric field. Therefore, an angle of the reflected light dark portion6 that causes the plasmon resonance forms a surface plasmon resonancecurve similar to the one obtained in the absence of applied electricfield.

As a specific example in which the frequency characteristics of asurface plasmon resonance angle are obtained, an example in which thephase of external vibration is compared with the phase of a signalcomponent of external vibration included in reflected light, so that thepoint of inflection of the frequency characteristics of a ligand, willbe described below. FIGS. 6(a) to 6(d) are diagrams for explaining therelationship between external vibration and binding of a receptor and aligand in the present invention. FIG. 6( a) is a diagram illustrating atemporal change in swept external vibration obtained by a frequencydivider. FIG. 6( b) is a diagram illustrating a temporal change in theamount of a ligand-receptor complex on a surface of a metal thin filmand upper electrode. FIG. 6( c) is a diagram illustrating a sum of thephase of applied external vibration and the phase of reflected lightthat causes plasmon resonance, and a temporal change in an output of adetector. FIG. 6( d) is a diagram illustrating a digital signal that isobtained by conversion of an analog signal of FIG. 6( c) using an A/Dconverter.

As illustrated in FIG. 6( c), a temporal change in the sum of the phaseof applied external vibration and the phase of reflected light thatcauses surface plasmon resonance is measured. A point of inflection of acurve obtained in this case is a point of inflection of frequencycharacteristics. For example, as the binding rate is increased to 10%,12%, and 14%, the frequency at the point of inflection is decreased tof1, f2, and f3. Therefore, by measuring a temporal change in the pointof inflection of the frequency characteristics, the degree of progressof binding of a ligand and a receptor can be measured.

The present inventors infer that the results are caused by the followingmechanism. When the external vibration 13 is applied to the receptor 9immobilized on the metal thin film and upper electrode 7, the receptor 9tries to follow the external vibration. In a low frequency region, thereceptor 9 can follow the phase of the external vibration 13 withoutdelay, and the dielectric constant of an evanescent region is changed,so that an angle that causes surface plasmon resonance has a width. Asthe frequency of the external vibration is further increased, thereceptor 9 follows the phase of the external vibration 13 with the delaygradually increased, and eventually cannot follow the phase of theexternal vibration 13. When the receptor 9 does not follow the phase ofthe external vibration 13, the dielectric constant of the evanescentregion on the metal thin film and upper electrode 7 becomes constant, sothat there is only a single angle that causes surface plasmon resonance.In other words, in the apparatus of FIG. 1, at a time when delay occursin the following, there occurs a deviation between the phase of theexternal vibration 13 and the phase of the reflected light 5 generatedby surface plasmon resonance, so that the amplitude of a combined waveof the phase of the external vibration 13 and the phase of the reflectedlight 5 that causes the plasmon resonance decreases. The point ofinflection of the frequency characteristics corresponds to a time atwhich delay occurs in the following. As the amount of a ligand-receptorcomplex increases, the mass exposed to external vibration increases, sothat a frequency (a frequency at the point of inflection) that does notfollow is reduced (see FIGS. 6( a) and 6(b)).

By converting an analog signal of the amplitude of the output combinedwave into a digital signal using the A/D converter 18 and detecting thedigital signal, it is possible to obtain a time until the point ofinflection of the frequency characteristics. Based on times t1, t2, andt3 until the point of inflection, the frequency characteristics can beused to detect the amount of a ligand binding to the receptor 9, whichconventionally is detected based on an angle that causes plasmonresonance (see FIG. 6( d)).

Although the apparatus that uses electrical vibration has beenheretofore described, a similar principle can be applied to an apparatusthat uses magnetic vibration or mechanical vibration.

Second Embodiment

In a second embodiment, another preferable embodiment of the apparatusof the present invention will be described. In FIG. 7, 51 indicates alight source. 52 indicates a receptor immobilized on a metal film andupper electrode. 53 indicates a ligand. 54 indicates a light receivingapparatus. 56 indicates a metal thin film and electrode. 58 indicates areflector. 59 indicates an electrode. 60 indicates an alternatingcurrent source. 61 indicates a frequency divider that divides afrequency of the alternating current source 60. 62 and 66 each indicatean active filter that passes a specific frequency. 55 indicates anexternal vibration. 64 indicates a capacitor. 65 indicates an amplifier.67 indicates a detector that compares a phase (θ₀) of external vibrationand a phase (θ₁) of a signal component of external vibration included inreflected light. 68 indicates an A/D converter that converts an analogsignal obtained by the detector 67 into a digital signal. 70 indicatesmeasuring light and 71 indicates reflected light.

The exemplary apparatus of the present invention of FIG. 7 is the sameas the exemplary apparatus of the present invention of FIG. 1, exceptthat the metal thin film and electrode 56 is provided in place of themetal thin film and upper electrode 7 and the electrode 59 is providedin place of the lower electrode 8. Further, the measuring light 70 isreflected on a surface of the metal thin film and electrode 56 aplurality of times by means of the reflector 58. The light source 51 andthe light receiving apparatus 54 are arranged so that the reflectedlight 71 is received by the light receiving apparatus 54. With such anarrangement, surface plasmon resonance is generated a plurality of timesin the apparatus of the present invention. As a result, the amplitude ofthe surface plasmon resonance angle is amplified, thereby making itpossible to achieve high-sensitivity measurement. In addition, even whenthe molecular weight of a receptor or a ligand is low, and therefore,the amplitude of a surface plasmon resonance angle is minute, theapparatus can amplify the amplitude of the surface plasmon resonanceangle, resulting in high-sensitivity measurement.

INDUSTRIAL APPLICABILITY

As described above, the analyzing method and apparatus of the presentinvention measure the frequency characteristics of a surface plasmonresonance angle with respect to external vibration, which makes itpossible to perform high-precision analysis without being affected byvibration and optical design. Therefore, the analyzing method andapparatus of the present invention are useful for analysis of a ligandin a sample, and are useful in the fields of, for example, biology,medicine, pharmacology, agriculture, and the like.

1. A method for analyzing a ligand in a sample, comprising the steps of:causing a sample containing a ligand and a metal thin film to contacteach other, wherein a receptor that can bind specifically to a ligand isimmobilized on one side of the metal thin film, an optical prism isprovided on an opposite side of the metal thin film, and the metal thinfilm can cause surface plasmon resonance, so that the ligand in thesample binds to the receptor; irradiating the side of the metal thinfilm opposite to the side on which the receptor is immobilized withmeasuring light using irradiating means for irradiating with measuringlight; receiving reflected light of the measuring light reflected on theside of the metal thin film using light receiving means for receivingreflected light of the measuring light; and detecting a change in asurface plasmon resonance angle caused by a change in a dielectricconstant of a vicinity of the metal thin film, based on the reflectedlight, using analyzing means for analyzing a ligand binding to thereceptor; further comprising applying external vibration to the side ofthe metal thin film on which the receptor is immobilized, using applyingmeans for applying external vibration to a region in which the receptoris immobilized, while irradiating the metal thin film with the measuringlight using the irradiating means; and obtaining frequencycharacteristics of a surface plasmon resonance angle with respect toexternal vibration using the analyzing means, and based on the frequencycharacteristics, analyzing a ligand in the sample binding to thereceptor, and wherein the analyzing means further includes comparingmeans for comparing a phase of the external vibration with a phase of asignal component of the external vibration included in the reflectedlight, and the step of obtaining the frequency characteristics comparesthe phase of the external vibration with the phase of the signalcomponent of the external vibration included in the reflected light,using the comparing means, to detect a point of inflection of thefrequency characteristics.
 2. The method according to claim 1, whereinat least one of a receptor and a ligand is charged.
 3. The methodaccording to claim 1, wherein the applying means is means for applyingat least one of electrical vibration, magnetic vibration, and mechanicalvibration.
 4. The method according to claim 1, wherein the applyingmeans is means for applying at least electrical vibration, and theanalyzing means further includes analyzing a physical property of theligand from the reflected light.
 5. The method according to claim 1,wherein an amount of a ligand in the sample binding to the receptor isanalyzed.
 6. The method according to claim 1, further comprising:detecting a degree of binding of the receptor and the ligand bymeasuring temporal change in the point of inflection of the frequencycharacteristics using the measuring means for measuring a temporalchange in the point of inflection of the frequency characteristics,wherein the analyzing means further includes comparing means forcomparing a phase of the external vibration with a phase of a signalcomponent of the external vibration included in the reflected light, andthe step of obtaining the frequency characteristics compares the phaseof the external vibration with the phase of the signal component of theexternal vibration included in the reflected light, using the comparingmeans, to detect a point of inflection of the frequency characteristics.7. The method according to claim 1, further comprising: causing thereflected light of the measuring light reflected on the side of themetal thin film on which the receptor is immobilized, using opticalmeans for causing the reflected light of the measuring light reflectedon the side of the metal thin film on which the receptor is immobilized,to impinge on the side further a plurality of times, wherein thereflected light of the measuring light received by the light receivingmeans is the reflected light of the measuring light reflected aplurality of times on the side of the metal thin film using the opticalmeans.
 8. The method according to claim 1, wherein a combination of areceptor and a ligand is an antigen and an antibody, an antibody and anantigen, a hormone and a hormone receptor, a hormone receptor and ahormone, a polynucleotide and a polynucleotide receptor, apolynucleotide receptor and a polynucleotide, an enzyme inhibitor and anenzyme, an enzyme and an enzyme inhibitor, an enzyme substrate and anenzyme, or an enzyme and an enzyme substrate.
 9. An apparatus foranalyzing a ligand in a sample, comprising: a metal thin film, wherein areceptor that can bind specifically to a ligand is immobilized on oneside of the metal thin film, an optical prism is provided on an oppositeside of the metal thin film, and the metal thin film can cause surfaceplasmon resonance; irradiating means for irradiating with measuringlight; light receiving means for receiving reflected light of themeasuring light reflected on the side of the metal thin film; analyzingmeans for analyzing a ligand binding to the receptor; wherein theapparatus further comprises applying means that can apply externalvibration to the side of the metal thin film on which the receptor isimmobilized, and a side of the metal thin film opposite to the side onwhich the receptor is immobilized, can be irradiated with measuringlight using the irradiating means while applying external vibrationusing the applying means, and the analyzing means can detect a change ina surface plasmon resonance angle from the reflected light and obtainfrequency characteristics of a surface plasmon resonance angle withrespect to external vibration, and based on the frequencycharacteristics, analyze a ligand in the sample binding to the receptorwherein the analyzing means further comprises comparing means forcomparing a phase of the external vibration with a phase of a signalcomponent of the external vibration included in the reflected light sothat a point of inflection of the frequency characteristics can bedetected.
 10. The apparatus according to claim 9, wherein at least oneof a receptor and a ligand is charged.
 11. The apparatus according toclaim 9, wherein the applying means is means for applying at least oneof electrical vibration, magnetic vibration, and mechanical vibration.12. The apparatus according to claim 9, wherein the applying means ismeans for applying at least electrical vibration, and the analyzingmeans further can analyze a physical property of the ligand from thereflected light.
 13. The apparatus according to claim 9, wherein anamount of a ligand in the sample binding to the receptor is analyzed.14. The apparatus according to claim 9, further comprising measuringmeans for measuring a temporal change in the point of inflection of thefrequency characteristics so that a degree of binding of the receptorand the ligand can be detected.
 15. The apparatus according to claim 9,further comprising optical means that can cause the reflected light ofthe measuring light reflected on the side of the metal thin film onwhich the receptor is immobilized, to impinge on the side further aplurality of times, wherein the reflected light of the measuring lightreceived by the light receiving means is the reflected light of themeasuring light reflected a plurality of times on the side of the metalthin film using the optical means.
 16. The apparatus according to claim9, wherein a combination of a receptor and a ligand is an antigen and anantibody, an antibody and an antigen, a hormone and a hormone receptor,a hormone receptor and a hormone, a polynucleotide and a polynucleotidereceptor, a polynucleotide receptor and a polynucleotide, an enzymeinhibitor and an enzyme, an enzyme and an enzyme inhibitor, an enzymesubstrate and an enzyme, or an enzyme and an enzyme substrate.